Comparison of gene expression profiles of human dental pulp cells treated with mineral trioxide aggregate and calcium hydroxide

Yong-Beom Kim; Won-Jun Shon; Woocheol Lee; Kee-Yeon Kum; Seung-Ho Baek; Kwang-Shik Bae

doi:10.5395/JKACD.2011.36.5.397

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Basic Research Comparison of gene expression profiles of human dental pulp cells treated with mineral trioxide aggregate and calcium hydroxide: Yong-Beom Kim, DDS, MSD, PhD¹, Won-Jun Shon, DDS, MSD, PhD², Woocheol Lee, DDS, MSD, PhD², Kee-Yeon Kum, DDS, MSD, PhD², Seung-Ho Baek, DDS, MSD, PhD², Kwang-Shik Bae, DDS, MSD, PhD^2,*; Journal of Korean Academy of Conservative Dentistry 2011;36(5):397-408.
DOI: https://doi.org/10.5395/JKACD.2011.36.5.397
Published online: September 14, 2011

¹Program in Conservative Dentistry, Seoul National University Graduate School

²Department of Conservative Dentistry, Seoul National University School of Dentistry and Dental Research Institute, Seoul, Korea

*Correspondence to: Kwang-Shik Bae, DDS, MSD, PhD., Professor, Department of Conservative Dentistry, Seoul National University School of Dentistry, Dental Research Institute, 28-2 Yongon-dong, Jongno-gu, Seoul, Korea 110-749, Tel, +82-2-2072-2651; Fax, +82-2-2072-3859; E-mail, baeks@snu.ac.kr

• Received: July 20, 2011 • Revised: August 17, 2011 • Accepted: August 21, 2011

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Abstract
REFERENCES

Abstract

Objectives:
This study investigated changes in gene expressions concerning of differentiation, proliferation, mineralization and inflammation using Human-8 expression bead arrays when white Mineral Trioxide Aggregate and calcium hydroxide-containing cement were applied in vitro to human dental pulp cells (HDPCs).
Materials and Methods:
wMTA (white ProRoot MTA, Dentsply) and Dycal (Dentsply Caulk) in a Teflon tube (inner diameter 10 mm, height 1 mm) were applied to HDPCs. Empty tube-applied HDPCs were used as negative control. Total RNA was extracted at 3, 6, 9 and 24 hr after wMTA and Dycal application. The results of microarray were confirmed by reverse transcriptase polymerase chain reaction.
Results:
Out of the 24,546 genes, 43 genes (e.g., BMP2, FOSB, THBS1, EDN1, IL11, COL10A1, TUFT1, HMOX1) were up-regulated greater than two-fold and 25 genes (e.g., SMAD6, TIMP2, DCN, SOCS2, CEBPD, KIAA1199) were down-regulated below 50% by wMTA. Two hundred thirty nine genes (e.g., BMP2, BMP6, SMAD6, IL11, FOS, VEGFA, PlGF, HMOX1, SOCS2, CEBPD, KIAA1199) were up-regulated greater than two-fold and 358 genes (e.g., EDN1, FGF) were down-regulated below 50% by Dycal.
Conclusions:
Both wMTA and Dycal induced changes in gene expressions related with differentiation and proliferation of pulp cells. wMTA induced changes in gene expressions related with mineralization, and Dycal induced those related with angiogenesis. The genes related with inflammation were more expressed by Dycal than by wMTA. It was confirmed that both wMTA and Dycal were able to induce gene expression changes concerned with the pulp repair in different ways.
Keywords: Calcium hydroxide; Microarray; Mineral trioxide aggregate; Pulp capping; Pulp cell; Reverse transcriptase polymerase chain reaction

Figure 1.

Classification of expressed genes by wMTA treatment. The genes which were up-or down-regulated by at least twofold following a cellular treatment with wMTA at any time point were classified using the panther classification system (http://www.pantherdb.org).

Figure 2.

Classification of expressed genes by Dycal treatment. The genes which were up-or down-regulated by at least twofold following a cellular treatment with Dycal at any time point were classified using the panther classification system.

Table 1.

Primer sequence list used in RT-PCR

Target gene	Forward	Reverse	Product Size (bp)
GAPDH	5′-GAA GTG GAA GGT CGG AGT C-3′	5′-GAA GAT GGT GAT GGG ATT TC-3′	313
BMP2	5′-ACT TTT GGA CAC CAG GTT GG-3′	5′-GCT GTT TGT GTT TGG CTT GA-3′	296
IL11	5′-GCA ATC TGA GGT CAC TGC AA-3′	5′-TCA CAG GGT GAC TTG TGG AA-3′	300
EDN1	5′-TTC CGT ATG GAC TTG GAA GC-3′	5′-TCT CCG ACC TGG TTT GTC TT-3′	300
CEBPD	5′-AGA AGT TGG TGG AGC TGT CG-3′	5′-CCA GGC TGT AGC TTC TTT GC-3′	300
FOSB	5′-GGC TCA ACA AGG AAA AAT GC-3′	5′-GAG GCC AGA AAT TCC AAT CA-3′	297
TUFT1	5′-GCA GAA AGA GGC AGA AGT CG-3′	5′-CCA AGC AGA ACG AGA AAA GG-3′	152
FOS	5′-CTT CCT GTT CCC AGC ATC AT-3′	5′-GTC AGA GGA AGG CTC ATT GC-3′	298
THBS1	5′-CCT GTG CTG CAG ACA TTG AT-3′	5′-GAA CAC AAT CCG GAT CAG CT-3′	148
DCN	5′-GGA CCG TTT CAA CAG AGA GG-3′	5′-AAT GCC ATC TTC GAG TGG TC-3′	147
BMP6	5′-TTC GTG CTG GAG TTT TGT TG-3′	5′-CTA ACC CTG TGG CGT GGT AT-3′	298
FGF5	5′-GCA AGT GCC AAG TTC ACA GA-3′	5′-AGG TGC AGA AAG GGG AAT CT-3′	303

Table 2.

Summary of expression profiles of genes differentially regulated by wMTA and Dycal

Fold change		Number	Gene symbol
wMTA	Dycal	Number	Gene symbol
fold > 2	fold > 2	17	NUAK1, TRIB1, SGK, FZD8, NOX4, ABCA1, SLC35F2, BPGM, IL11,
			PMEPA1, ASNS, RCAN1, BMP2, PTGS2, PTHLH, STC1, HMOX1
	fold < -2	1	EDN1
	2 > fold > -2	25	LOH3CR2A, ANGPTL4, SH3BP5L, MMP10, DENR, DACT1, SCD, SLC46A3, UAP1L1, PDPN, FOSB, TUFT1, TNS1, ZNF365, ESM1, HBEGF, NKX3-1, MAZ, AK3L1, ETV4, MT1X, FOLR3, LRRC17, THBS1, COL10A1
	Subtotal	43
fold < -2	fold > 2	7	KIAA1199, DHRS3, CEBPD, KYNU, SMAD6, ATOH8, SOCS2
	fold < -2	5	PDE5A, HERC4, DDIT4L, VCAM1, SLC25A22
	2 > fold > -2	13	TIMP2, FBN2, COL3A1, MFGE8, CNTNAP1, MFAD4, UBA7, PRRX2, VPS24, SIPA1, ASAM, PHF15, DCN
	Subtotal	25

Table 3.

The relative mRNA expression levels of genes analyzed by RT-PCR

Symbol	6hr		9hr		24hr
Symbol	wMTA	Dycal	wMTA	Dycal	wMTA	Dycal
BMP2	1.88	2.31
IL11			1.99	2.38
EDN1					1.41	0.26
CEBPD					1.16	2.07
FOSB			1.28
TUFT1			1.57
FOS				1.77
THBS1					1.89
DCN					0.58
BMP6						11.93
FGF5						0.25

The relative level of gene expression was normalized against GAPDH messenger RNA, and the control was set as 1.0. Optical density values represent the mean (n=3).

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Comparison of gene expression profiles of human dental pulp cells treated with mineral trioxide aggregate and calcium hydroxide

J Korean Acad Conserv Dent. 2011;36(5):397-408. Published online September 14, 2011

DOI: https://doi.org/10.5395/JKACD.2011.36.5.397

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Figure

Comparison of gene expression profiles of human dental pulp cells treated with mineral trioxide aggregate and calcium hydroxide

Figure 1. Classification of expressed genes by wMTA treatment. The genes which were up-or down-regulated by at least twofold following a cellular treatment with wMTA at any time point were classified using the panther classification system (http://www.pantherdb.org).

Figure 2. Classification of expressed genes by Dycal treatment. The genes which were up-or down-regulated by at least twofold following a cellular treatment with Dycal at any time point were classified using the panther classification system.

Figure 1.

Figure 2.

Comparison of gene expression profiles of human dental pulp cells treated with mineral trioxide aggregate and calcium hydroxide

Target gene	Forward	Reverse	Product Size (bp)
GAPDH	5′-GAA GTG GAA GGT CGG AGT C-3′	5′-GAA GAT GGT GAT GGG ATT TC-3′	313
BMP2	5′-ACT TTT GGA CAC CAG GTT GG-3′	5′-GCT GTT TGT GTT TGG CTT GA-3′	296
IL11	5′-GCA ATC TGA GGT CAC TGC AA-3′	5′-TCA CAG GGT GAC TTG TGG AA-3′	300
EDN1	5′-TTC CGT ATG GAC TTG GAA GC-3′	5′-TCT CCG ACC TGG TTT GTC TT-3′	300
CEBPD	5′-AGA AGT TGG TGG AGC TGT CG-3′	5′-CCA GGC TGT AGC TTC TTT GC-3′	300
FOSB	5′-GGC TCA ACA AGG AAA AAT GC-3′	5′-GAG GCC AGA AAT TCC AAT CA-3′	297
TUFT1	5′-GCA GAA AGA GGC AGA AGT CG-3′	5′-CCA AGC AGA ACG AGA AAA GG-3′	152
FOS	5′-CTT CCT GTT CCC AGC ATC AT-3′	5′-GTC AGA GGA AGG CTC ATT GC-3′	298
THBS1	5′-CCT GTG CTG CAG ACA TTG AT-3′	5′-GAA CAC AAT CCG GAT CAG CT-3′	148
DCN	5′-GGA CCG TTT CAA CAG AGA GG-3′	5′-AAT GCC ATC TTC GAG TGG TC-3′	147
BMP6	5′-TTC GTG CTG GAG TTT TGT TG-3′	5′-CTA ACC CTG TGG CGT GGT AT-3′	298
FGF5	5′-GCA AGT GCC AAG TTC ACA GA-3′	5′-AGG TGC AGA AAG GGG AAT CT-3′	303

Fold change		Number	Gene symbol
wMTA	Dycal	Number	Gene symbol
fold > 2	fold > 2	17	NUAK1, TRIB1, SGK, FZD8, NOX4, ABCA1, SLC35F2, BPGM, IL11,
			PMEPA1, ASNS, RCAN1, BMP2, PTGS2, PTHLH, STC1, HMOX1
	fold < -2	1	EDN1
	2 > fold > -2	25	LOH3CR2A, ANGPTL4, SH3BP5L, MMP10, DENR, DACT1, SCD, SLC46A3, UAP1L1, PDPN, FOSB, TUFT1, TNS1, ZNF365, ESM1, HBEGF, NKX3-1, MAZ, AK3L1, ETV4, MT1X, FOLR3, LRRC17, THBS1, COL10A1
	Subtotal	43
fold < -2	fold > 2	7	KIAA1199, DHRS3, CEBPD, KYNU, SMAD6, ATOH8, SOCS2
	fold < -2	5	PDE5A, HERC4, DDIT4L, VCAM1, SLC25A22
	2 > fold > -2	13	TIMP2, FBN2, COL3A1, MFGE8, CNTNAP1, MFAD4, UBA7, PRRX2, VPS24, SIPA1, ASAM, PHF15, DCN
	Subtotal	25

Symbol	6hr		9hr		24hr
Symbol	wMTA	Dycal	wMTA	Dycal	wMTA	Dycal
BMP2	1.88	2.31
IL11			1.99	2.38
EDN1					1.41	0.26
CEBPD					1.16	2.07
FOSB			1.28
TUFT1			1.57
FOS				1.77
THBS1					1.89
DCN					0.58
BMP6						11.93
FGF5						0.25

Table 1. Primer sequence list used in RT-PCR

Table 2. Summary of expression profiles of genes differentially regulated by wMTA and Dycal

Table 3. The relative mRNA expression levels of genes analyzed by RT-PCR

The relative level of gene expression was normalized against GAPDH messenger RNA, and the control was set as 1.0. Optical density values represent the mean (n=3).